Higher eukaryotic gene regulation is a complex process that is poorly understood at the mechanistic level. Improper regulation of steroid responses can result in a wide range of diseases and affect tumor development and progression. An unanswered question in the steroid receptor field is how the different steroid receptors differentially regulate genes while recognizing nearly identical DNA sequences. The promoters of steroid-controlled genes often contain multiple response element binding sites, thereby enabling cooperative interactions between adjacently bound receptors. This proposal addresses the functional connection between cooperativity and differential gene regulation using the glucocorticoid and progesterone receptors as a model system. The hypothesis of this proposal is that the extent to which GR and PR activate a gene correlates with the extent of GR- and PR- specific cooperative interactions when assembling on the promoter of that gene. Aim 1 will focus on a thermodynamic dissection of the microscopic binding energetics that define the interaction between the glucocorticoid receptor and HRE2 promoter. Quantitative footprints and statistical mechanical analyses will be used to carry out these Aims. The cellular relevance of these receptor- specific energetic differences measured in vitro will be analyzed in Aim 2 using ChIP assays and quantitative RT-PCR. Aim 3 will extend this study to GR and PR natural promoters by measuring in vitro composite binding energetics cellular measurements of promoter occupancies.